Project Summary/Abstract Telomeres are a required feature of eukaryotic linear chromosomes that serve to distinguish chromosome ends from DNA damage, and consist of long repeating sequences of double-stranded and single-stranded DNA. Shelterin is responsible for protecting telomere ends from the DNA-damage response (DDR) pathway. Shelterin is crucial to cellular health, and functional defects are linked to premature aging, genetic disorders, and cancer. Despite shelterin?s important roles in genome maintenance, little is known about the mechanism by which it protects telomeres. Shelterin is composed of six different proteins, which assemble in a hierarchical manner and robustly interact in vitro. It requires most components for telomere end protection, and individual knock-outs are typically lethal. Shelterin is remodels telomere ends into a ?t-loop? structure. While components of shelterin have been pinpointed as having DNA-remodeling capabilities, the molecular basis of how shelterin accomplishes this is enigmatic. One of the key requirements to elucidating shelterin?s function, and the overall goal of these studies, rests in determining the details of shelterin?s structural features and to examine shelterin?s molecular interactions with DNA. The proposed research will achieve this goal using an interdisciplinary approach involving biochemistry, computational modeling, and single-particle EM. Thus far in my postdoctoral career in the Nogales lab at UC Berkeley, I have obtained training in high- resolution cryo-EM structure determination of helical filaments known as microtubules. Moving forward, I plan to focus on studying the role of shelterin in binding DNA and mediating telomere end protection using single-particle negative stain EM and cryo-EM. To achieve these goals, I propose to: (1) Determine the architecture of shelterin using negative stain EM, (2) use cryo-EM to determine the mechanism of single-stranded DNA protection, and (3) use cryo-EM to examine the molecular basis of shelterin?s DNA remodeling abilities. During the K99 training period, I will apply biochemical tools to optimize recombinant shelterin for EM imaging and I will use single-particle EM approaches to visualize, for the first time, the structure of shelterin and the details of shelterin-DNA interactions. I will use this information in the R00 period to build upon what I?ve learned by studying the compositional variability of shelterin and how it affects shelterin structure and function. I believe that the mentorship and strong background of Eva Nogales and Ahmet Yildiz together with the training support provided by the K99/R00 award will allow me to build a strong foundation to enable my success as an independent investigator while illuminating the molecular mechanism of shelterin?s function. The results of the proposed studies will be to elucidate shelterin?s molecular mechanism in binding telomere DNA. This will lead to new hypotheses that can be tested functionally, and an understanding of how shelterin-DNA interactions contributes to telomere end structure that can be exploited for future therapeutics.